Project Summary: Single-Molecule Electronic Nucleic Acid Sequencing-by-Synthesis Using Novel Tagged Nucleotides and Nanopore Constructs With past NIH funding, we developed a single-molecule real-time electronic nanopore-based sequencing-by- synthesis system (Nanopore-SBS). We reported on the method?s ability to generate DNA sequencing reads at single- molecule level with single-base resolution. The method relies on sequencing complexes embedded in a lipid membrane, consisting of a highly processive polymerase tethered to an ?-hemolysin nanopore, bound to a DNA template and primer. Each complex is individually addressable by electrodes of an integrated circuit array chip designed by our collaborators at Genia (Roche). Addition of the 4 nucleotides, each with a different polymeric tag on its terminal phosphate, initiates the polymerase sequencing reaction. In the time between binding a tagged nucleotide by polymerase and its incorporation, the tag is drawn into the nanopore and partially interrupts ionic current through the pore. Four tags are designed such that each reduces the current by a different amount, allowing the sequence to be determined in real time. While the Nanopore-SBS approach already produces good quality sequences, further optimization and development are needed to increase sequencing accuracy, while maintaining the capability of our nanopore-based single-molecule electronic system to produce long reads in real time. In this proposal, our established team of chemists, molecular biologists, and biochemists will develop new classes of tagged nucleotides and modified polymerase-pore assemblies, to achieve desired polymerase catalytic rates and more efficient and consistent tag capture by the pores. We will use high ratios of unincorporable-to-incorporable tagged nucleotides to perform Nanopore-SBS. This will provide ample time to register currents due to the 4 unique tags on the unincorporable A, C, G and T nucleotides which display template-dependent binding to the polymerase ternary complex but are not incorporated into the growing DNA strand, followed by a new current level due to a 5th tag on the incorporable nucleotide which serves to mark the transition to the extension step. This effectively eliminates insertion and deletion artifacts in the sequence, increasing accuracy, and will be especially advantageous in homopolymer repeat regions of the DNA. This approach allows detection of a single nucleotide binding event multiple times (stutters) before the actual incorporation event, overcoming the inherent limitation of single molecule detection methods that only allow one chance for measurement. Modifications of the nanopore will achieve even more discrete tag signatures, further enhancing the method?s accuracy. After optimizing the system with synthetic DNA templates, circular DNA libraries will be generated from bacterial and viral genomes to test the sequencing approach. With the improved tagged nucleotides, better regulated reaction kinetics, and newly designed polymerase-pore complexes, we will test the accuracy of our system on the nanopore arrays by sequencing these libraries at high coverage and comparing the results with other sequencing systems.